This invention relates to a process for the analysis of the isoenzyme pattern of an enzyme in biological fluids, extracts or excretions.
Isoenzymes are molecular variants of the same enzyme which differs in molecular structure generally by only a few amino acid groups. Since the relative distribution of isoenzymes differs in different organs and since, like ordinary enzymes, isoenzymes are released into the bloodstream or other body fluids when an organ is diseased or damaged, the identity of the isoenzymes which appear is indicative of the organ from which they originate. For example, the enzyme lactic dehydrogenase occurs in at least five isoenzyme forms which are characteristically found in varying amounts in different organs; the isoenzyme patterns can be employed to diagnose over a dozen abnormal conditions, e.g., heart attacks and liver diseases. Similarly, creatine phosphokinase isoenzyme patterns can be used to detect minor heart damage undetectable by electrocardiograms.
Because all of the isoenzyme forms of a particular enzyme are structurally very similar, highly specific electrophoretic or immunological methods are required to identify and quantify individual isoenzyme forms.
German Patent Application No. P 21 28 670.4, corresponding to copending, commonly assigned U.S. patent application Ser. No. 261,215, filed June 9, 1972, now U.S. Pat. No. 3,932,221, the contents of which are incorporated by reference herein, describes an immunological test using practically completely precipitating antisera, obtained by means of pure human isoenzyme antigens (homologous antisera), to conduct an organ-, tissue- and/or disease-specific analysis of the isoenzyme pattern of an enzyme occurring in multiple molecular forms in human body fluids, extracts or excretions.
The isoenzyme test described therein has a number of advantages; in principle the activities of the individual isoenzymes of an eneyzme occurring in multiple molecular forms can be specifically determined in a quantitative manner. Due to the simplicity of the process, it has become possible to detect the entire isoenzyme patterns of diagnostically interesting enzymes without great expenditues in time and material by means of a routine analysis. Another advantage of this process is the precision of the method, surpassing frequently the accuracy of the heretofore employed isoenzyme determination methods, e.g., the various electrophoretic methods. At the same time, this process frequently makes it possible at all for the first time to clearly differentiate between the individual isoenzyme and/or hybrids.
Because of the breadth of possibilities of application and due to the simplicity with which it can be carried out, the method provides flawless differentiation of the isoenzyme pattern of an enzyme occurring in multiple molecular forms, so that indications can be made of the organ specificity and, if the isoenzymes of a certain enzyme are distributed in the cell among various cellular compartments, also of the intracellular distribution. Such analyses have heretofore been impractical due to the technical expenditures, complexity, and inaccuracy of earlier known methods.
The aforementioned isoenzyme test, however, also entails certain difficulties, primarily connected with the fact that the isoenzyme antigen required to produce the necessary antisera must first be isolated for human body parts. This causes severe problems for the antisera manufacturer on an ethical and also on a practical level.
Since the antigens employed for the production of the required antisera stem from human biological materials, the antisera manufacturer must work up autopsy material. Although heterologous antisera can also be obtained from animals, the use of human antigens for the manufacture thereof nonetheless represents a serious burden. Additionally, the working up of cadaver materials in order to obtain the antigen is problematical for the persons executing this task. Objectively, there is also the danger of infection, especially with infectious hepatitis, when working up the human liver.
Further practical difficulties arise because autopsy material can normally be taken only after a certain period of time, e.g. 18 hours after death. During this time, sensitive organs and/or tissues, e.g., the brain, are already subjected to autolysis. Therefore, it is extremely difficult to obtain pure isoenzyme-antigen preparations.
Faced with these difficulties, the question arose whether or not it would be possible to employ animal tissue matter for the isolation of the required isoenzyme antigons. However, the following prejudices exist against this procedure:
The precipitin reaction is known to be extremely specific. Even minute changes in the structure of an antigen can lead to the production of an antiserum resulting in reduced precipitation with the unchanged antigen, or even no precipitation at all. It is also known that analogous human and animal isoenzymes differ from one another to various degrees with respect to their structures.
Taking these facts into account, and in view of the consideration that, for the described isoenzyme test, a practically quantitatively removal of one or more isoenzyme antigens and thus maximum specificity of the antisera employed are required, it could be foreseen that the results attainable with heterologous antisera against animal antigens would not satisfy the specificity requirements given by the use of homologous antisera as the standard.